This invention relates to factors which interact with CD11b/CD18 integrin complex or the I-domain portion of CD11b/CD18 integrin complex and inhibit leukocyte activity. These factors inhibit neutrophil activity, including inhibition of neutrophil activation and adhesion of neutrophils to vascular endothelial cells. These factors also inhibit eosinophil activity, including inhibition of eosinophil adhesion to vascular endothelial cells.
Leukocytes are a class of cells comprised of lymphocytes, monocytes and granulocytes. The lymphocytes include within their class, T-cells (as helper T-cells and cytotoxic or suppressor T-cell), B-cells (as circulating B-cells and plasma cells), third population or natural killer (NK) cells and antigen-presenting cells. Monocytes include within their class, circulating blood monocytes, Kupffer cells, intraglomerular mesangial cells, alveolar macrophages, serosal macrophages, microglia, spleen sinus macrophages and lymph node sinus macrophages. Granulocytes include within their class, neutrophils, eosinophils, basophils, mast cells, (as mucosa-associated mast cells and connective tissue mast cells).
Neutrophils are an essential component of the host defense system against microbial invasion. In response to soluble inflammatory mediators released by cells at the site of injury, neutrophils emigrate into tissue from the bloodstream by crossing the blood vessel wall. At the site of injury, activated neutrophils kill foreign cells by phagocytosis and by the release of cytotoxic compounds, such as oxidants, proteases and cytokines. Despite their importance in fighting infection, neutrophils themselves can promote tissue damage. During an abnormal inflammatory response, neutrophils can cause significant tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Alternatively, neutrophils that stick to the capillary wall or clump in venules may produce tissue damage by ischemia. Such abnormal inflammatory responses have been implicated in the pathogenesis of a variety of clinical disorders including adult respiratory distress syndrome (ARDS); ischemia-reperfusion injury following myocardial infarction, shock, stroke, and organ transplantation; acute and chronic allograft rejection; vasculitis; sepsis; rheumatoid arthritis; and inflammatory skin diseases (Harlan et al., 1990 Immunol. Rev. 114, 5).
Neutrophil adhesion at the site of inflammation is believed to involve at least two discrete cell-cell interactive events. Initially, vascular endothelium adjacent to inflamed tissue becomes sticky for neutrophils; neutrophils interact with the endothelium via low affinity adhesive mechanisms in a process known as xe2x80x9crollingxe2x80x9d. In the second adhesive step, rolling neutrophils bind more tightly to vascular endothelial cells and migrate from the blood vessel into the tissue.
Neutrophil rolling along affected vascular segments and other initial low affinity contacts between neutrophils and the endothelium are reported to be mediated by a group of monomeric, integral membrane glycoproteins termed selecting. All three of the selectins so far identified, that is L-selectin (LECAM-1 or LAM-1) present on the surface of neutrophils, E-selectin (endothelial leukocyte adhesion molecule-1 or ELAM-1) present on endothelial cells and P-selectin (granule membrane protein-140, GMP-140, platelet activation-dependent granule-external membrane protein, PADGEM or CD62) expressed on endothelial cells, have been implicated in neutrophil adhesion to the vascular endothelium (Jutila et al., 1989 J. Immunol 143, 3318; Watson et al., 1991 Nature 349, 164; Mulligan et al., J. Clin. Invest. 88, 1396; Gundel et al., 1991; J. Clin. Invest. 88, 1407; Geng et al., 1990 Nature 343, 757; Patel et al., 1991 J. Cell Biol. 112, 749). The counter-receptor for E-selectin is reported to be the sialylated Lewis X antigen (sialyl-Lewisx) that is present on cell-surface glycoproteins (Phillips et al., 1990 Science 250, 1130; Walz et al., 1990 Science 250, 1132; Tiemeyer et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 1138; Lowe et al., 1990 Cell 63, 475). Receptors for the other selectins are also thought to be carbohydrate in nature but remain to be elucidated.
The more stable secondary contacts between neutrophils and endothelial cells are reported to be mediated by a class of cell adhesion molecules known as integrins. Integrins comprise a broad range of evolutionarily conserved heterodimeric transmembrane glycoprotein complexes that are present on virtually all cell types. Members of the leukocyte-specific CD18 (xcex22) family of integrins, which include CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1, Mo-1 or CR3) have been reported to mediate neutrophil adhesion to the endothelium (See Larson and Springer, 1990 Immunol Rev. 114, 181). Endothelial cell counter-receptors for these integrins are the intercellular cell adhesion molecules ICAM-1 and ICAM-2 for CD11a/CD18 and ICAM-1 for CD11b/CD18 respectively (Rothlein et al., 1986 J. Immunol. 137, 1270; Staunton et al., 1988 Cell 52, 925; Staunton et al., 1989 Nature 339, 61). The ICAMs are monomeric transmembrane proteins that are members of the immunoglobulin superfamily.
The CD11b/CD18 integrin is expressed on a variety of leukocytes, including monocytes, macrophages, granulocytes, large granular lymphocytes (NK cells), and immature and CD5+ B cells (Kishimoto, T. K., Larson, R. S., Corbi, A. L., Dustin, M. L., Staunton, D. E., and Spriger, T. A. (1989) Adv. in Immunol. 46,149-182). CD11b/CD18 has been implicated in a variety of leukocyte functions including adhesion of neutrophils to endothelial cells (Prieto, J., Beatty, P. G., Clark, E. A., and Patarroyo, M. (1988) Immunology 63, 631-637; Wallis, W. J., Hickstein, D. D., Schwartz, B. R., June, C. H., Ochs, H. D., Beatty, P. G., Klebanoff, S. J., and Harlan, J. M. (1986) Blood 67, 1007-1013; Smith, C. W., Marlin, S. D., Rothlein, R., Toman, C., and Anderson, D. C. (1989) J. Clin. Invest. 83, 2008-2017) and release of hydrogen peroxide from neutrophils (Shappell, S. B., Toman, C., Anderson, D. C., Taylor, A. A., Entman, M. L. and Smith, C. W. (1990) J. Immunol. 144, 2702-2711; Von Asmuth, E. J. U., Van Der Linden, C. J., Leeuwenberg, J. F. M., and Buurman, W. A. (1991) J. Immunol. 147,3869-3875). This integrin may play a role in neutrophil and monocyte phagocytosis of opsonized (i.e. C3bi-coated) targets (Beller, D. I., Springer, T. A., and Schreiber, R. D. (1982) J.Exp. Med. 156,1000-1009). It has also been reported that CD11b/CD18 contributes to elevated natural killer activity against C3bi-coated target cells (Ramos, O. F., Kai, C., Yefenof, E., and Klein, E. (1988) J. Immunol. 140,1239-1243).
The activation of endothelial cells and neutrophils is believed to represent an important component of neutrophil-mediated inflammation. Factors that induce cell activation are termed agonists. Endothelial cell agonists, which are believed to include small regulatory proteins such as tumor necrosis factor (TNFxcex1) and interleukin-1xcex1 (IL-1xcex1), are released by cells at the site of injury. Activation of endothelial cells has been reported to result in the increased surface expression of ICAM-1 (Staunton et al., 1988 Cell 52, 925) and ELAM-1 (Bevilacqua et al., 1987 Proc. Natl. Acad. Sci. (USA) 84, 9238). Raised levels of expression of these adhesive molecules on the surface of activated endothelial cells is believed to lead to the observed increased adhesivity of neutrophils for the vascular endothelium near sites of injury.
Activation of the neutrophil results in profound changes to its physiological state, including shape change, ability to phagocytose foreign bodies and release of cytotoxic substances from intracellular granules. Moreover, activation is believed to greatly increase the affinity of adhesive contacts between neutrophils and the vascular endothelium, perhaps through a conformational change in the CD11b/CD18 integrin complex on the neutrophil surface (Vedder and Harlan, 1988 J. Clin. Invest. 81, 676; Buyon et al., 1988 J. Immunol. 140, 3156). Factors that have been reported to induce neutrophil activation include IL-1xcex1, GM-CSF, G-CSF, MIP-1, IL-8 (IL-8=interleukin-8, GM-CSF=granulocyte/monocyte-colony stimulating factor, G-CSF=granulocyte-colony stimulating factor), TNFxcex1, the complement fragment C5a, the microbe-derived peptide formyl-Met-Leu-Phe and the lipid-like molecules leukotriene B4 (LTB4) and platelet activating factor (Fuortes and Nathan, 1992, in Molecular Basis of Oxidative Damage by Leukocytes Eds Jesaitis, A. J. and Dratz, E. A. (CRC Press) pp. 81-90). In addition, phorbol esters (e.g., phorbol 12-myristate 13-acetate; PMA) have been proposed as a potent class of synthetic lipid-like neutrophil agonists. With the exception of PMA, these agonists are believed to activate neutrophils by binding receptors on their surface. Receptors that are occupied by agonist molecules are believed to initiate within the neutrophil a cascade of events that ultimately will result in the physiological changes that accompany neutrophil activation. This process is known as signal transduction. The lipid-like PMA is proposed to affect neutrophil activation by passing through the plasma membrane at the cell surface and directly interacting with intracellular components (i.e., protein kinase) of the signal transduction machinery.
There exist two general classes of compounds that have been reported to down regulate the function of neutrophils, and these compounds have been shown to mitigate inflammation. One group of anti-inflammatory compounds has been proposed to function as inhibitors of neutrophil activation, and presumably adhesion, by acting on components of the signal transduction machinery. A second class of anti-inflammatory compounds has been proposed to block neutrophil infiltration into inflammatory foci by acting as direct inhibitors of the adhesive receptors that mediate contact between neutrophils and the vascular endothelium.
Many of the anti-inflammatory compounds currently used as therapeutics, including prostaglandins, catecholamines, and a group of agents known as non-steroidal anti-inflammatory drugs (NSAIDs), are believed to fall into the first category (Showell and Williams, 1989, in Immunopharmacology, eds. Gilman, S. C. and Rogers, T. J. [Telford Press, NJ] pp 23-63). For example, the enhanced adhesiveness observed for TNFxcex1-activated neutrophils has been reported as associated with decreased levels of a mediator of signal transduction, cyclic AMP (cAMP). (See Nathan and Sanchez, 1990 JCB 111, 2171). Exposure of neutrophils to prostaglandins and catecholamines has been correlated with elevated levels of intracellular cyclic AMP (Showell and Williams, 1989). While signal transduction inhibitors have been used extensively as anti-inflammatory therapeutic agents, they have been shown to have several disadvantages including poor efficacy in acute inflammatory conditions, lack of specificity and undesirable side-effects such as gastric or intestinal ulceration, disturbances in platelet and central nervous system function and changes in renal function (Insel, 1990 in The Pharmacological Basis of Therapeutics, eds. Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P. [Pergamon, N.Y.], 8th Ed., pp. 638-681).
Glucocorticoids have long been recognized for their anti-inflammatory properties. Steroid induced inhibition of neutrophils has been reported for several neutrophil functions, including adherence (Clark et al., 1979 Blood 53, 633-641; MacGregor, 1977 Ann. Intern. Med. 86, 35-39). The mechanisms by which glucocorticoids modulate neutrophil function are not well understood, but they are generally believed to involve the amplification or suppression of new proteins in treated neutrophils that play a key role in the inflammatory process (Knudsen et al., 1987 J. Immunol. 139, 4129). In particular, a group of proteins known as lipocortins, whose expression is induced in neutrophils by glucocorticoids, has been associated with anti-inflammatory properties (Flower, 1989 Br. J. Pharmacol. 94, 987-1015). Lipocortins may exert anti-neutrophil effects by interacting with sites on the neutrophil surface (Camussi et al., 1990 J. Exp. Med. 171, 913-927), but there is no evidence to suggest that the lipocortins act by directly blocking adhesive proteins on the neutrophil. Apart from their beneficial anti-inflammatory properties, glucocorticoids have been associated with significant side-effects. These include suppression of pituitary-adrenal function, fluid and electrolyte disturbances, hypertension, hyperglycemia, glycosuria, susceptibility to infection, ulcers, osteoporosis, myopathy, arrest of growth and behavioral disturbances (Insel, 1990)
A second class of anti-inflammatory compounds which are reported as direct inhibitors of neutrophil adhesion to the vascular endothelium are monoclonal antibodies. Monoclonal antibodies that recognize and block ligand-binding functions of some of these adhesive molecules have been reported to act as in vivo inhibitors of neutrophil-mediated inflammation. In particular, monoclonal antibodies to the CD18 subunit of the CD18 integrin complexes (i.e., CD11a/CD18, CD11b/CD18 and CD11c/CD18) on the surface of neutrophils have been reported to prevent a variety of neutrophil-mediated tissue injury in animal models, including pulmonary edema induced by reperfusion (Horgan et al, 1990 Am. J. Physiol. 259, L315-319), organ injury induced by hemorrhagic shock (Mileski et al, 1990 Surgery 108, 206-212), myocardial damage following ischemia/reperfusion (Winquist et al, 1990 Circulation III-701), edema and tissue damage following ischemia/reperfusion of the ear (Vedder et al, 1990 Proc. Natl. Acad. Sci. (USA) 87, 2643-2646), brain edema and death produced by bacterial meningitis (Tuomanen et al, 1989 J. Exp. Med. 170, 959-968), vascular injury and death in endotoxic shock (Thomas et al, 1991 FASEB J. 5, A509) and indomethacin-induced gastric injury (Wallace et al, 1991 Gastroenterology 100, 878-883).
Monoclonal antibodies directed to the CD11b subunit have been reported by Todd, R. F. et al., U.S. Pat. No. 4,840,793 (Jun. 20, 1989), Todd, R. F. et al., U.S. Pat. No. 4,935,234 (Jun. 19, 1990), Schlossman, S. F. et al., U.S. Pat. No. 5,019,648 (May 28, 1991) and Rusche, J. R. et al., International Application No. WO 92/11870 (Jul. 23, 1992). Monoclonal antibodies directed to CD18 subunit have been reported by Arfors, K. E., U.S. Pat. No. 4,797,277 (Jan. 10, 1989), Wright, S. D. et al., European Patent Application No. 346,078 (Dec. 13, 1989), Law, M. et al., European Patent Application No. 438,312 (Jul. 24, 1991), Law, M, et al., European Patent Application No. 440,351 (Aug. 7, 1991), Wright, S. D. et al., U.S. Pat. No. 5,147,637 (Sep. 15, 1992) and Wegner, C. D. et al., European Patent Application No. 507,187 (Oct. 7, 1992).
Antibodies to other adhesive molecules have also been reported to have anti-inflammatory properties. Monoclonal antibodies that recognize the counter-receptor of CD11a/CD18 and CD11b/CD18, ICAM-1 have been reported to prolong cardiac allograft survival (Flavin et al, 1991 Transplant. Proc. 23, 533-534) and prevent chemically induced lung inflammation (Barton et al, 1989 J. Immunol. 143, 1278-1282). Furthermore, anti-selectin monoclonal antibodies have also been reported as active in animal models of neutrophil-mediated inflammation. Monoclonal antibodies to L-selectin have been reported to prevent neutrophil emigration into inflamed skin (Lewinshon et al., 1987 J. Immunol. 138, 4313) and inflamed ascites (Jutila et al., 1989 J. Immunol. 143, 3318; Watson et al., 1991 Nature 349, 164). Reports have also described inhibition of neutrophil influx into inflamed lung tissue by anti E-selectin monoclonal antibodies (Mulligan et al., 1991 J. Clin. Invest. 88, 1396; Gundel et al., 1991 J. Clin. Invest. 88, 1407). While monoclonal antibodies to adhesive proteins have demonstrated the feasibility of using neutrophil adhesion inhibitors as anti-inflammatory agents, their utility as therapeutics requires further evaluation.
Soluble adhesive receptors obtained by genetic engineering have been proposed as anti-inflammatory, compounds. Soluble receptors, in which the transmembrane and intracellular domains have been deleted by recombinant DNA technology, have been tested as inhibitors of neutrophil adhesion to endothelial cells. The functional use of recombinant soluble adhesive molecules has been reported using CD11b/CD18 (Dana et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 3106-3110) and L-selectin (Watson et al., 1991).
Recently, a new class of anti-leukocyte compounds collectively termed xe2x80x9cleumedinsxe2x80x9d has been reported. These compounds have been reported to block the recruitment in vivo of T lymphocytes and neutrophils into inflammatory lesions. The mechanism of action of the leumedins is unclear, but there is evidence that they do not function by blocking neutrophil activation (Burch et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 355). It remains to be determined if leumedins block neutrophil infiltration by direct interference with adhesive molecules.
It has been suggested that parasites survive in their host by modulating host immunity and inflammatory response though the mechanisms by which this occurs remains unclear (Leid, W. S., 1987, Veterinary Parasitology, 25: 147). In this regard, parasite-induced immunosuppression in rodent models has been proposed (Soulsby et al., 1987, Immunol Lett. 16, 315-320). The various aspects of the modulation of host immunity by helminth parasites to evade immunological attack has recently been reviewed. See Maizels et al. (1993), Nature, 365:797-805.
Various parasites have been reported to have an affect on neutrophils of their host. For example, a protein isolated from the cestode, Taenia taeniaeformis, has been reported to inhibit chemotaxis and chemokinesis of equine neutrophils, as well as inhibit neutrophil aggregation (C. Suquet et al., 1984, Int""l J. Parasitol., 14: 165; Leid, R. W. et al., 1987, Parasite Immunology, 9: 195; and Leid, R. W. et. al., 1987, Int""l J. Parasitol., 17: 1349). Peritoneal neutrophils from mice infected with the cestode, Echinococcus multiocularis, have been reported to lose their ability to migrate toward parasite antigens and nonspecific chemoattractants with increasing time of infection (Alkarmi, T. et al., Exptl. Parasitol., 1989, 69: 16). The nematode, Trichinella spiralis, has been reported to either excrete and/or secrete factors which inhibit chemotaxis and p-nitroblue tetrazolium reduction (i.e., release of oxidative metabolites) but enhance chemokinesis of human neutrophils (Bruschi, F. et al., 1989, Wiadomosci Parazytologiczne, 35: 391). The sera of humans infected with the nematode, Trichinella spiralis, has been reported to inhibit leukocyte chemotaxis and phagocytosis (Bruschi, F. et al., 1990, J. Parasitol., 76: 577). The saliva of the tick, Ixodes dammini, has been reported to inhibit neutrophil function (Ribeiro et al, 1990, Exp. Parasitol., 70, 382). A protein secreted by the cestode, Echinococcus granulosus, has been reported to inhibit human neutrophil chemotaxis (Shepard, J. C. et al., 1991, Mol. Biochem. Parasitol., 44: 81).
Another component of the host defense mechanism against invading pathogens are eosinophils. Functionally, eosinophils are similar to neutrophils in that both cell types have the ability to phagocytose and to release compounds that are either directly or indirectly toxic to pathogenic organisms. Eosinophils are distinguished from neutrophils by their morphologic features, constituents, products and associations with specific diseases. Although eosinophils have been reported to be capable of killing bacteria in vitro, this class of leukocyte alone is not believed sufficient to defend against bacterial infections in vivo. Instead, it is thought that eosinophils afford primary defense against large organisms such as helminthic parasites (Butterworth A E, 1984; Adv. Parasitol. 23:143-235). Also, it is widely held that eosinophils can play a major role in certain inflammatory diseases. Specifically, substances released from eosinophils that are known collectively as cationic granule proteins, including major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin, have been implicated in asthma (Gleich G J and Adolphson, C R, 1986; Adv. Immunol. 39:177-253), inflammatory bowel disease (Hxc3xa4llren, R, 1989; Am. J. Med. 86:56-64) and atopic dermatitis (Tsuda, S, et al, 1992; J. Dermatol. 19:208-213). Moreover, other eosinophil products such as superoxide anions, hydroxyl radicals and singlet oxygen may also be involved in damage to host tissue in inflammatory disease states (Petreccia, DC et al, 1987, J. Leukoc. Biol. 41:283-288; Kanofsky, J R et al, 1988; J. Biol. Chem. 263:9692-9696).
An early step in eosinophil-mediated inflammatory disease is believed to be the movement of eosinophils from the vascular compartment to tissue. The first step in this extravasation process is reported to be the adherence of eosinophils to the luminal surface of the vascular endothelium. Although mechanisms of eosinophil-endothelial cell adhesion are not as well defined as those involving adhesion by neutrophils, it is reported that members of the CD11/CD18 family of integrins on the surface of the eosinophil are involved in eosinophil-endothelial adhesion (Lamas, A M, et al, 1988; J. Immunol. 140:1500; Walsh, G M, et al, 1990; Immunology 71:258), and it is reported that the endothelial cell counter-receptor is likely ICAM-1 (Wegner, C D, et al, 1990; Science 247:456-459). A second integrin known as VLA-4 (very late antigen-4, a4b1) that is present on eosinophils, lymphocytes and monocytes but not neutrophils, is thought to contribute to eosinophil adherence by binding to the VCAM-1 (vascular cell adhesion molecule-1) that is expressed on the surface of endothelial cells (Dobrina, A, et al, 1991, J. Clin. Invest. 88:20). IL-1 treatment of the endothelial cell monolayers has been reported to induce an increased adhesiveness for human basophils, eosinophils and neutrophils but treatment of these endothelial cells with an antibody directed to VCAM-1 was reported to inhibit both basophil and eosinophil adhesion but not neutrophil adhesion. It has also been reported that monoclonal antibodies against VCAM-1 inhibit lymphocyte and monocyte cell adhesion to stimulated endothelium (Carlos et al. (1990), Blood, 76:965-970 Rice et al., J. Exp. Med. (1990), 171:1369-1374) but not to neutrophils.
Approaches to the treatment of eosinophil-mediated inflammation have been similar to those adopted for neutrophil-mediated disease. For example, potential therapeutics under investigation for eosinophil-mediated inflammation include glucocorticoids (Evans, P M, et al., 1993, J. Allergy Clin. Immunol. 91:643-650). As is the case for other agents that have been reported to modulate neutrophil function, these agents have been found to be sub-optimal in that they are relatively non-specific and toxic. A second approach to anti-eosinophil therapy has been the use of compounds that directly inhibit the adhesion of eosinophils to vascular endothelium. It has been reported that in animal models of asthma, monoclonal antibody against ICAM-1 blocks eosinophil infiltration into tissues. Wegner et al. (1990), Science, 247:456-459. ICAM-1 and functional derivatives thereof have been proposed as anti-inflammatory agents. Anderson et al., European Patent Application No. 314,863 (Apr. 29, 1988); Wegner et al., International Application No. WO 90/10453 (Sep. 20, 1990)
However, there remains a need for potent, highly specific inhibitors of neutrophil and eosinophil function, in particular, adhesion to vascular endotbelium, as a treatment for abnormal granulocyte-mediated inflammation. The present invention describes potent and specific inhibitors of neutrophil and eosinophil activity, in particular the adhesion of these granulocytes to vascular endothelial cells, derived from hookworms (such as Ancylostoma caninum) and related species.
Among other factors, the present invention is based on our finding that the Neutrophil Inhibitory Factor of the present invention represents a pioneering step toward the development of a new generation of anti-inflammatory therapeutic products. This discovery will enable therapy for inflammatory disease based entirely on specific inhibition of the inflammatory response. The therapeutic advantages of this novel approach are realized through the specificity of Neutrophil Inhibitory Factor compared to current clinical treatment modalities such as steroids, catecholamines, prostaglandins, and nonsteroidal anti-inflammatory agents. The currently used therapeutic agents demonstrate poor efficacy and multiple adverse reactions due to generalized systemic effects that non-specifically target numerous biological processes in addition to the inflammatory process. Nonetheless, the existence of this extensive panel of anti-inflammatory agents, although suboptimal, and the total funds expended by the pharmaceutical industry in research in this area point to significant medical needs for effective anti-inflammatory agents and suggests that the novel and highly specific Neutrophil Inhibitory Factors of the present invention have important applications.
As noted in the Background, the inflammatory response may result in clinical syndromes ranging from debilitating arthritis and asthma to life threatening shock. In view of the severity of these disorders, the vast number of individuals afflicted therewith and the lack of suitable therapeutic intervention, the need for a breakthrough therapy represents a long felt need which has not been met. The Neutrophil Inhibitory Factor of the present invention is believed to meet this need by providing the potential for a lifesaving therapy which is currently being sought throughout the international medical and pharmaceutical research communities.
Further, in view of the myriad conditions associated with undesired and/or abnormal inflammatory conditions which appear to be associated with neutrophil activity, there remains a need for potent, highly specific inhibitors of neutrophil function, in particular, adhesion to vascular endothelium, as a treatment for abnormal neutrophil-mediated inflammation. The present invention is believed to fulfill this need by disclosing a potent and specific inhibitor of neutrophil activity, in particular the adhesion of neutrophils to vascular endothelial cells, derived from hookworms (such as Ancylostoma caninum) and related species.
The present invention is directed to a neutrophil inhibitory factor (xe2x80x9cNeutrophil Inhibitory Factorxe2x80x9d or xe2x80x9cNIFxe2x80x9d) which may be isolated from natural sources or made by recombinant methods. Neutrophil Inhibitory Factor is a protein which is neither an antibody, a member of the integrin or selectin families nor a member of the immunoglobulin superfamily of adhesive proteins and which when isolated from a parasitic worm is a glycoprotein. Recombinant NIF produced by certain expression systems may or may not be glycosylated, or may be glycosylated to a variable degree. However, such NIFs whether glycosylated or not are considered to be within the scope of the present invention.
In one aspect, the present invention provides NIFs which contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of
(a) Arg-X1-X2-Phe-Leu-X3 -X4-His-Asn-Gly-Tyr-Arg-Ser-X5-Leu-Ala-Leu-Gly-His-X6-X7-Ile, [SEQ. ID. NO. 1] wherein X1 is Leu or Arg; X2 is Gln, Lys or Arg; X3 is Ala or Arg; X4 is Leu or Met; X5 is Lys, Arg, Leu or Ile; X6 is Val or Ile; and X7 is Ser, Gly or Asn;
(b) Ala-X8-X9-Ala-Ser-X10-Met-Arg-X11-Leu-X12-Tyr-Asp-Cys-X13-Ala-Glu-X14-Ser-Ala-Tyr-X15-Ser-Ala, [SEQ. ID. NO. 2] wherein X8 is His or Pro; X9 is Thr, Arg or Ser; X10 is Arg or Lys; X11 is Ile or Tyr; X12 is Asp, Lys or Glu; X13 is Asp or Glu; X14 is Gly, Lys or Arg; and X15 is Glu, Met, Thr or Val;
(c) Ser-X16-Phe-Ala-Asn-X17-Ala-Trp-Asp-X18-Arg-Glu-Lys-X19-Gly-Cys-Ala-Val-Val-X20-Cys, [SEQ. ID. NO. 3] wherein X16 is Asn or Asp; X17 is Val or Leu; X18 is Ala or Thr; X19 is Leu, Val or Phe; and X20 is Thr, Lys or Asn;
(d) His-Val-Val-Cys-His-X21-X22-Pro-Lys, [SEQ. ID. NO. 41] wherein X21 is Tyr or Ile; and X22 is Gly or no residue;
(e) Ile-Tyr-X23-X24-Gly-X25-Pro-cys-X26-X27-Cys-X28-X29-Tyr, [SEQ. ID. NO. 5] wherein X23 is Thr, Ser, Lys or Glu; X24 is Thr, Val or Ile; X25 is Val, Lys or Thr; X26 is Arg, Ser or Asp; X27 is Asn, Gly, Asp or Arg; X28 is Asn, Ser or Thr; and X29 is Gly, Glu or Asp; and
(f) CyS-X30-X31-Asp-X32-Gly-Val-Cys-X33-Ile, [SEQ. ID. NO. 6] wherein X30 is His, Ile or Asn; X31 is Ala, Pro or Asp; X32 is Glu, Val, Asp or Ile; and X33 is Ile, Val or Phe. Such NIFs exhibit neutrophil inhibitory activity.
In another aspect, the present invention provides mutant NIFS wherein certain asparagine residues are replaced with glutamine residues which is believed to result in the reduced glycosylation of these NIFs. The mutant NIFS contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of peptides (a) to (f) hereinabove. Such NIFs exhibit neutrophil inhibitory activity.
In another aspect, the present invention provides NIFs which contain, as part of their total amino acid sequence, an amino acid sequence encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to certain nucleic acid probes. Such NIFs exhibit neutrophil inhibitory activity. The present invention includes within its scope these nucleic acid probes.
In another aspect, the present invention provides nucleic acid molecules encoding for a NIF and which are isolated as described herein. Such isolated nucleic acid molecules include expression vectors containing a nucleic acid sequence encoding a NIF. The present invention also includes the host cells transformed by such expression vectors.
In another aspect, the present invention provides methods for making biologically active NIFs, wherein such NIFs are expressed and, optionally, secreted. The present invention also includes the NIFs made by these methods.
In another aspect, the present invention provides methods of making NIFs comprising preparing a cDNA library from a source suspected of having a NIF and hybridizing certain oligonucleotide probes of the present invention to the nucleic acid molecules from the source. Such NIFs exhibit neutrophil inhibitory activity. The present invention also includes the NIFs made by these methods.
In another aspect, the present invention provides methods of detecting in a sample the presence of a nucleic acid molecule encoding a NIF, which methods comprise the combining of the sample thought to contain such nucleic acid molecule with a probe of the present invention and detecting the presence of hybridized probe.
In another aspect, the present invention provides monoclonal antibodies which bind to NIF. The present invention also includes the hybridoma cell lines which make such antibodies, a method of purifying NIF using such monoclonal antibodies and method of detecting in a sample the presence of NIF using such antibodies.
In another aspect, the present invention provides a method for detecting in a sample NIF mimics which compete with NIFs for binding to the CD11b/CD18 receptor, which method comprises contacting a sample with the CD11b/CD18 receptor. Also provided is a method for detecting in a sample NIF mimics which compete with NIFs for binding to the I-domain portion of the CD11b/CD18 receptor, which method comprises contacting a sample with a recombinant peptide comprising the I-domain of CD11b/CD18 receptor. Such NIF mimics exhibit neutrophil inhibitory activity. The present invention also includes the NIF mimics detected by these methods.
In another aspect, the present invention provides a method for detecting in a sample a NIF antagonist which prevents NIF binding to the CD11b/CD18 receptor, which method comprises contacting such sample with the CD11b/CD18 receptor. Also provided is a method for detecting in a sample NIF antagonist which compete with NIFs for binding to the I-domain portion of the CD11b/CD18 receptor, which method comprises contacting a sample with a recombinant peptide comprising the I-domain of CD11b/CD18 receptor. Such NIF antagonists do not exhibit neutrophil inhibitory activity themselves. The present invention also includes the NIF antagonists detected by these methods.
In another aspect, the present invention provides methods of using NIF to treat inflammatory conditions, especially to prevent or decrease inflammatory responses, which methods comprise administering to a mammal a therapeutically effective amount of NIF.
Other features and advantages of the present invention will be apparent from the following descriptions of the preferred embodiments and from the claims.
In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.
The term xe2x80x9camino acidxe2x80x9d refers to the natural L-amino acids. The natural amino acids shall be referred to by their names or may be abbreviated as shown below:
The term xe2x80x9camino acid residuexe2x80x9d refers to xe2x80x94NHxe2x80x94CH(R)xe2x80x94COxe2x80x94, wherein R is the side chain group distinguishing each amino acid. For cyclic amino acids, the residue is 
wherein x is 1, 2 or 3 representing the azetidine-carboxylic acid, proline or pipecolic acid residues, respectively.
The term xe2x80x9cisoformxe2x80x9d refers to a family of related proteins from a single organism having homologous sequences of amino acid residues interspersed with variable sequences.
The term xe2x80x9cnucleic acidxe2x80x9d refers to polymers of either deoxynucleic acids or ribonucleic acids, either single-stranded or double-stranded.
The term xe2x80x9cisolated nucleic acidxe2x80x9d refers to nucleic acids which are isolated by biochemical or molecular biology techniques such as centrifugation, chromatography, electrophoresis, hybridization and the like.
The term xe2x80x9cNIF mimicxe2x80x9d refers to a small molecule, peptide, peptide analog or protein, which competes with NIF for binding to the CD11b/CD18 receptor or the I-domain portion of the CD11b/CD18 recptor. A NIF mimic is also characterized as having neutrophil inhibitory activity, eosinophil inhibitory activity or both such activities.
The term xe2x80x9cNIF antagonistxe2x80x9d refers to a small molecule, peptide, peptide analog or protein, which prevents the binding of NIF to the CD11b/CD18 receptor or the I-domain portion of this receptor, and does not possess any significant neutrophil inhibitory activity. A NIF antagonist prevents binding of NIF to the CD11b/CD18 receptor or the I-domain portion of the CD11b/CD18 receptor, by binding to a site on NIF which is required for binding to the receptor in effect sterically hindering binding, or alternatively, by binding to a site on NIF which results in a conformational change to the site needed for such binding which change substantially weakens or abolishes binding.